Genome sequencing projects have rapidly accelerated the pace of gene discovery and have led to the identification of thousands of new genes of higher order organisms, including humans. The challenge ahead is to identify the biological functions of many of the newly discovered genes. DNA microarray—also known as “gene-chip”—technology has emerged as a powerful tool for genome-wide analysis of gene expression and gene-sequence variations. One caveat of microarray technology is that protein abundance within the cell does not always correlate with expressed mRNA levels. Because the function of a gene is directly related to the activity of its translated protein, an alternative and possibly superior approach to elucidate gene functions lies in direct analysis of the functions of the specific proteins for which the gene encodes.
The current prevailing approach for analyzing protein function in vivo is to employ cell-based assays. These types of assays are used to study the function of one particular gene in a cellular context, through gene transfection and protein delivery. For the gene transfection approach, cells are transfected with a vector containing a specific gene that leads to the overexpression of the gene product. With regard to the protein delivery approach, cells are “transfected” with a functional protein, including antibodies, using membrane-disrupting, pore-forming reagents or other reagents, such as liposomes, as a carrier to deliver the protein across the cell membrane. Using a variety of functional assays, the effects of introduced DNA or proteins on cellular physiology are then detected.
Protein delivery, i.e., protein transduction is the process by which a peptide or protein motif crosses the cell plasma membrane. Traditionally, methods to introduce antibodies, peptides or other membrane-impermeable molecules into cells include micro-injection and electroporation. The obvious disadvantages of these techniques are that they tend to be toxic to the recipient cells, they are non-specific (i.e., anything can enter or exit the cell once the membrane is disrupted), and they exhibit low transfection efficiency and substantial variability. To overcome the disadvantage associated with these techniques, researchers have developed a number of protein-transduction domains (PTDs) that mediate protein delivery into cells. These PTDs or signal peptide sequences are naturally occurring polypeptides of 15 to 30 amino acids, which normally mediate protein secretion in the cells. They are composed of a positively charged amino terminus, a central hydrophobic core and a carboxyl-terminal cleavage site recognized by a signal peptidase. Recently, researchers have shown that a number of membrane-translocating peptides can successfully mediate delivery of polypeptides, protein domains, and full-length protein, including antibodies into cells using solution-based protein transfection protocols. Recently, researchers have also demonstrated the use of lipid liposomes or the like for protein delivery.
Traditionally, however, these approaches have been limited since they are solution-based formats. Only one gene or protein may be studied per assay. As more there are more than 35,000 genes present in the human genome, for instance, and approximately 10,000 of these genes are expressed as proteins in any given cell type, a high-throughput method for studying gene function is needed.